Targeted Testing

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Overall, the analytical sensitivity of this test is expected to be high because the great majority of variants reported to date in the ALDOB gene are detectable via DNA sequencing. Santer and colleagues (2005) reported two causative variants in 93% of the 72 HFI patients they studied, and one causative variant in the remaining 7%, for an overall detection rate of ~96.5% (139 out of 144 alleles carried causative variants). Their cohort of patients consisted primarily of individuals of German and Mediterranean heritage. In 2008, Davit-Spraul and colleagues reported the identification of two causative variants in 96.5% of individuals in a group of 92 HFI patients. A single causative variant was identified in the remaining 3.5% of patients, for an overall detection rate of ~98.4% (181 out of 184 alleles carried causative variants). Approximately three-fourths of the patients in their cohort were French, with the remaining being of Belgian or Mediterranean heritage.

A small number of patients have been shown to carry large, exonic deletions that are undetectable via sequencing, which may account for the remaining unidentified causative variants in these studies.

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Clinical Features

Hereditary fructose intolerance (HFI) is a congenital condition that is characterized by abdominal pain, vomiting and hypoglycemia shortly after the ingestion of fructose, sucrose, or sorbitol. Effects during infancy can be severe and become apparent either upon the initiation of weaning from breastfeeding or upon the ingestion of fructose-containing infant formula. Feeding difficulties and overall failure to thrive also often accompany the primary symptoms in infants. In undiagnosed individuals, continued ingestion of the toxic sugars can lead to further growth retardation, hepatic and renal injury, and can eventually lead to liver and kidney failure, coma and death, especially in small infants. Surviving infants and children typically develop strong aversions to foods that induce symptoms and have far lower rates of dental caries than the general population as a result of their decreased sugar consumption. However, even this strong food aversion is not typically sufficient to eliminate all foods containing noxious sugars from the diet, and thus affected individuals may suffer from episodic symptoms throughout life, although the effects generally become less severe with age. In some patients, hepatomegaly and elevated liver transaminases occur when fructose sources are not completely eliminated from the diet. If an individual remains undiagnosed, IV-infusions containing fructose, sucrose or sorbitol may cause life-threatening complications (Ali et al. 1998; Cos 1994; Steinmann et al. 2014).

Dietary exclusion of fructose, sucrose and sorbitol (including that found in medicines) results in the elimination of symptoms and complete recovery, if diagnosis is made early enough. Individuals who completely eliminate the noxious sugars from their diet are anticipated to have a normal life expectancy (Ali et al. 1998; Cos 1994; Steinmann et al. 2014).

Genetics

Hereditary fructose intolerance is an autosomal recessive disorder, and ALDOB is the only gene in which defects are known to cause HFI. To date, approximately 60 causative variants have been reported in the ALDOB gene (Steinmann et al. 2014; Human Gene Mutation Database; HFI Mutational Database). About one-third of reported pathogenic variants are missense, with the remainder being nonsense, splicing and small insertions and deletions. Two variants upstream of the coding sequence, c.-214G>A and c.-11+1G>C, have also been reported (Coffee and Tolan 2010). The variants are spread throughout the ALDOB gene, with no known mutational hotspots (HFI Mutational Database). Three variants (A150P, A175D and N335K) are known to be the most common pathogenic variants and, in fact, have been shown to account for upwards of 60% of HFI cases in European and North American individuals (Cross et al. 1990; Davit-Spraul et al. 2008; Santer et al. 2005). Most other variants are rare or private mutations. A relatively small percentage of cases (~3-5%) have been attributed to larger exonic deletions that are generally undetectable via standard Sanger sequencing (Human Gene Mutation Database; Cross and Cox 1990; Ferri et al. 2012).

Hereditary Fructose Intolerance is caused by catalytic defects in the aldolase B protein, also known as fructose-1,6-bisphosphate aldolase B. Two other isozymes, aldolase A and aldolase C, are found in humans. The tissues in which these isozymes predominate differ from aldolase B, which is expressed mainly in the liver, kidneys and small intestine. Aldolase B catalyzes the conversion of fructose-1-phosphate to D-glyceraldehyde and dihydroxyacetone phosphate, as well as the conversion of fructose-1,6-bisphosphate to D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. It is thought that the clinical symptoms observed in aldolase B deficient individuals are due to both the buildup of the fructose-1-phosphate and fructose-1,6-bisphosphate precursors, as well as due to the decreased amount of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate available to continue through the glycolytic pathway (Ali et al. 1998; Cos 1994; Steinmann et al. 2014).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of the ALDOB gene plus ~10 bp of flanking non-coding DNA on each side. Additionally, ALDOB gene sequencing includes the regions encompassing the documented variants c.-214G>A and c.-11+1G>C. This test is performed in two tiers.Tier 2 is performed if Tier 1 results are negative, and involves sequencing the aforementioned non-coding regions as well as the remaining exons (2-4, 6-8). We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known variants or to confirm research results.

Indications for Test

Individuals with a positive intravenous fructose load test or aldolase B enzymatic assay results from liver or intestinal biopsy samples are good candidates for this test, as are those exhibiting clinical symptoms suggestive of hereditary fructose intolerance. Family members of patients who have known ALDOB variants are candidates. We will also sequence the ALDOB gene to determine carrier status.

TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.